Apparatus for the preparation of objects for microscopic examination

ABSTRACT

Apparatus for the preparation of resin-impregnated specimens for microscopic examination, comprising a stack of embedding boxes 62 each containing a specimen. A variety of reagents required for the preparation of the specimens are selectively fed to the stack of embedding boxes by a delivery pump 13. The reagent fed to the stack of embedding boxes is shaked by a reagent shaker mechanism 14 for ready infiltration into the specimens.

TECHNICAL FIELD

This invention relates to apparatus for the preparation of objects forstudy under the electron and other type of microscope in medical andother fields of science, such that specimens such as those extractedfrom living matter are automatically fixed and embedded in resinmaterial with use of minimal amounts of reagents.

BACKGROUND ART

Objects of microscopic examination in medical and other fields ofscience require pretreatment with reagent and embedment or infiltrationof resins into the specimens for solidification. The embedded specimensare sectioned, and the sections are mounted on glass slides for opticalmicroscopy and on mesh grids for electron microscopy.

Heretofore, for automatically embedding objects as above stated,apparatus has been used which comprises a plurality (e.g. 20) of reagentand resin containers, each in the form of an upstanding, open-top,bottomed cylinder, in annular arrangement. Embedding boxes containingobjects of microstopic study have been dipped successively in thereagent and resin containers. The embedding boxes have been suspendedfrom the periphery of a disc which is centrally supported by a rotary,vertically reciprocable post disposed at the center of the annular rowof reagent and resin containers. The disk is moved up and down androtated at intervals for dipping the embedding boxes in the successivecontainers.

The micrologist has, however, experienced some inconveniences with theforegoing prior art apparatus. Objects of microscopic study to beembedded are as small in size as from 0.5 by 0.5 millimeters (mm) to 1.0by 1.0 mm. The embedding boxes for immersing such objects in requiredreagents are each approximately 10 mm in diameter. Nevertheless, fromabout 20 to 30 cubic centimeters (cc) of each reagent is required forprocessing a batch of, say, 20 objects. The used reagents must bediscarded, and fresh supplies of reagents must be used for processinganother batch. However, since some reagents are expensive, it isuneconomical to discard the considerable amounts of them each time onebatch of objects is treated.

Another weakness of the prior art is that the embedding boxes holdingthe specimens are merely immersed in the liquid. This conventionalmethod fails to realize ready infiltration of the liquid into thespecimens.

A further drawback of the prior art manifests itself when, in the courseof embedment, the specimens are immersed in alcohol of progressivelyhigher concentrations, from about 50% up to 100%. The known apparatushas necessitated the provision of a series of five separate containersfor 50%, 70%, 80%, 95% and 100% alcohol. Alcohol preparations of 50% and100% concentrations are available on the market, but the micrologist hashad to take the trouble of himself preparing the other concentrations.

The present invention aims at the elimination of all the foregoinginconveniences.

DISCLOSURE OF THE INVENTION

The apparatus for the preparation of objects for microscopic examinationin accordance with the invention comprises a reagent select mechanismfor selectively communicating a plurality of reagent receptacles,containing reagents for the embedment of specimens, with a deliveryconduit having a delivery pump capable of bidirectional rotation and ofblocking the flow of any selected reagent therethrough when out ofrotation, and an embedding box holder for holding a stack of embeddingboxes each in the form of a short tube having a mesh bottom for holdingthereon a specimen to be embedded. The embedding boxes are stacked withtheir peripheries in fluid-tight engagement with each other. Theembedding box holder has a bottom fitting for communicative connectionwith one end of the delivery conduit, and a top fitting forcommunicative connection with one end of an overflow conduit leading toa waste receptacle. The waste receptacle communicates with one ofseveral ports of the reagent select mechanism via a waste conduit.

Upon loading of the embedding boxes, containing the specimens to beembedded, on the embedding box holder, the delivery pump is to be setinto operation for feeding a selected reagent from one of the reagentreceptacles into the embedding boxes within the holder via the deliveryconduit, thereby flooding the specimens within the embedding boxes withthe reagent. The delivery pump is to be held out of motion during thesubsequent immersion of the specimens in the reagent. The reagent selectmechanism is actuated at the same time to establish communicationbetween the delivery conduit and waste conduit while the delivery pumpis out of motion.

Upon completion of the specimen immersion for a required length of time,the delivery pump may be set into rotation in a reverse direction fordischarging the used reagent into the waste receptacle.

Following the discharge of the used reagent, the reagent selectmechanism may be re-actuated to place the delivery conduit incommunication with another reagent receptacle containing the nextreagent to be supplied. Then the delivery pump may again be set intorotation in a forward direction for delivering the next selected reagentinto the embedding boxes.

The foregoing cycle of operation may be repeated for embedding thespecimens within the embedding boxes. For treating the specimens withalcohol of successively higher concentrations, the lowest concentration(50%) alcohol may first be pumped into the embedding boxes. Then, withthis lowest concentration alcohol left undischarged, the highestconcentration (100%) alcohol may be pumped into the embedding boxesthereby gradually increasing the alcohol concentration therein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the general organization of the apparatus for thepreparation of objects for microscopic examination in accordance withthe invention;

FIG. 2 is a top plan view of the reagent select mechanism;

FIG. 3 is a side elevation, partly in section, of the reagent selectmechanism;

FIG. 4 is a side elevation of a shaker mechanism;

FIG. 5 is a top plan view, partly in section, of the shaker mechanism;

FIG. 6 is a top plan view of the embedding box holder;

FIG. 7 is a sectional view of the embedding box holder taken along theline A--A of FIG. 6; and

FIG. 8 is an elevation of the embedding box holder as seen from theright hand side of FIG. 7.

BEST MODE FOR CARRYING OUT THE INVENTION

With reference to FIG. 1, a diagrammatic illustration of the generalorganization of the apparatus in accordance with the invention, there isprovided at 1 a reagent housing which is provided with partitions 2 and3 for dividing its interior into three chambers 4, 5 and 6. Each ofthese chambers accommodates one or more reagent receptacles 7a, 7b and7c. The reagent receptacles 7a in the chamber 5 contain reagents such asglutaraldehyde, sodium acetate, etc., which are being held at arelatively low temperature of, for example, 4° C. The reagentreceptacles 7b in the chamber 5 contain, for example, a 50% alcoholsolution and 100% alcohol at room temperature. The single reagentreceptacle 7c in the chamber 6 contains, for example, a resin at atemperature of approximately 37° C.

Inserted respectively in the reagent receptacles 7a, 7b and 7c aresuction conduits 8 having their open bottom ends held close to thebottoms of the reagent receptacles. The suction conduits 8 communicatethe reagent receptacles 7a , 7b and 7c with respective female couplingmembers 10 of a reagent select mechanism 9. In addition to the femalecoupling members 10, which are held in fixed locations, the reagentselect mechanism 9 comprises a single male coupling member 11 movableinto fluid-tight engagement with any selected one of the female couplingmembers.

A delivery conduit 12 has its lower end communicatively affixed to themale coupling member 11 and extends upwardly therefrom. The deliveryconduit 12 is provided both with a delivery pump 13 for forcing arequired reagent up through the delivery conduit from the male couplingmember 11, and with a shaker mechanism 14 for imparting vibration to thereagent being delivered. The upper end of the delivery conduit 12 iscommunicatively coupled to the bottom end of an embedding box holder 15in which is held a stack of embedding boxes containing objects ofmicroscopic examination to be embedded. The delivery pump 13 should bebidirectional and, when out of motion, should block the flow of theliquid therethrough. The shaker mechanism 14 operates, when the deliverypump 13 is out of motion following the delivery of a desired reagen intothe embedding case holder 15, to agitate or shake the reagent on thedownstream side of the shaker mechanism and hence to assure intimatecontact of the reagent with the specimens in the embedding cases.

The embedding box holder 15 has its top end communicatively coupled toan overflow conduit 17. The embedding box holder 15 communicates withthe overflow conduit 17 via an enlargement 16 providing a space intowhich part of the reagent on the downstream side of the shaker mechanism14 can flow during the above described operation of the shakermechanism.

The overflow conduit 17 communicates with a waste receptacle 18, whichmay be contained in the chamber 4 of the reagent housing 1. Alsocommunicating with the waste receptacle 18 is an air intake conduit 20having an air filter 21 on its end away from the waste receptacle. Theair intake conduit 20 is further provided with a three-way valve 19 forselectively placing the waste receptacle 18 in and out of communicationwith the air intake conduit 20 and with an exhaust conduit 23. A vacuumpump 22 is provided to the exhaust conduit 23 for the discharge ofexhaust gases via an activated charcoal filter 24. The waste receptacle18 further communicates by way of a waste liquid conduit 25 with one ofthe female coupling members 10 of the reagent select mechanism 9.Notwithstanding the showing of FIG. 1, the air filter 21 is dispensable,and the conduit 20 may be used for communicating the waste receptacle 18with the activated charcoal filter 24 or with the reagent receptacles.

Given hereafter is a more detailed discussion of the reagent selectmechanism 9, shaker mechanism 14 and embedding box holder 15 which areincorporated in the micrological apparatus of the foregoing generalconfiguration in accordance with the invention.

As illustrated in detail in FIGS. 2 and 3, the reagent select mechanism9 comprises a table 26 of circular shape. Rigidly mounted on theunderside of the table 26 in alignment therewith is an electric drivemotor 27 having a drive shaft 28 coupled to a rotary post 29 which isrotatably supported over the table. A solenoid 30 is secured to one sideof the rotary post 29 at or adjacent the top end therof. The dependingplunger 31 of the solenoid 30 is coupled to one end of an uprightconnecting rod 32, the other end of which is secured to a reciprocator34. This reciprocator is movable up and down as guided by a guide 33secured to the rotary post 29 in an underlying relation to the solenoid30. A helical compression spring 35 acts between guide 33 andreciprocator 34 for biasing the latter downwardly. The reciprocator 34has rigidly mounted to its bottom end a plug 37 having its bottom endportion formed into frustoconical shape to serve as the noted malecoupling member 11, FIG. 1, of the reagent select mechanism 9. Inconstant communication with the delivery conduit 12, FIG. 1, a pipe 36extends through the plug 37.

Rigidly mounted to the table 26 are a plurality of socket members 38which correspond to the female coupling members 10, FIG. 1, of thereagent select mechanism 9 and which are arranged in an annular rowalong the periphery of the table 26 in concetric relation thereto. Aswill be seen by referring back to FIG. 1, each socket member 38 hascoupled thereto one of the suction conduits 8, which are incommunication with the reagent receptacles 7a, 7b and 7c, or the wasteliquid conduit 25 in communication with the waste receptacle 18.

FIG. 4 and 5 show the details of the shaker mechanism 14. It takes theform of a gripper mechanism 39 which operates to periodically grip andsqueeze a hose 40 of elastic or pliant material forming a part of thedelivery conduit 12, FIG. 1. The gripper mechanism 39 comprises a pairof L-shaped gripping jaws 44 having a pair of teeth 43 which aredisposed on opposite sides of the hose 40 and which are periodicallyactuated toward and away from each other by intermittent energization ofa solenoid 42 on a standard 41. The pair of gripping jaws 44 are mountedon upstanding pins 45 for pivotal motion in a horizontal plane. Thesolenoid 42 has a plunger 46 coupled to a link 47 which has a flange 47aon its end away from the solenoid. The flange 47a engages the pair ofgripping jaws 44 in such a manner that upon energization of the solenoid42, the retracting plunger 46 causes the gripping jaws 44 to jointlypivot in opposite directions, resulting in the movement of the pair ofteeth 43 toward each other. A pair of helical compression springs 49 aremounted respectively between the gripping jaws 44 and a fixed support 48on the standard 41, biasing the gripping jaws in such directions thatthe pair of teeth 43 tend to travel apart from each other.

The embedding box holder 15 is illustrated in detail in FIGS. 6-8. Seenat 50 is an upstanding baseplate fabricated from metal, typicallyaluminum, that is a good conductor of heat. The baseplate 50 has ahorizontal ledge 51 formed on its bottom end. A pair of threaded studs52 are erected on the ledge 51 with a spacing therebetween. Also mountedto the ledge 51, in a position intermediate the pair of studs 52, is abottom fitting 53 extending downwardly therefrom for communicativeconnection of the top end of the delivery conduit 12 to the embeddingbox holder 15.

Extending upwardly from the baseplate ledge 51, the pair of threadedstuds 52 have their top end portions inserted in and through holes 55defined in a yoke 54 of generally T-shaped vertical section in thevicinities of its opposite ends. Nuts 56 are fitted over the top ends ofthe studs 52 protruding upwardly of the yoke 54. The yoke 54 has athickened midportion 57 with a fluid passageway 58 defined verticallytherethrough. A top fitting 59 is firmly inserted in this passageway 58from above for communicative connection of one end of the overflowconduit 17, FIG. 1, to the embedding box holder 15.

A plurality of substantially annular box carriers 63, each having anembedding box 62 mounted fast therein, are stacked, via a pair of holderplates 60 and 61, between the baseplate ledge 51, having the lowerfitting 53, and the yoke 54 having the top fitting 59. The box carriers63 are so shaped as to be firmly stacked up, and O-ring seals 65 arefitted in annular grooves 64 formed in their top surfaces to seal thejoints between the box carriers against the leakage of the reagent. Theembedding boxes 62 within their carriers 63 are each in the shape of ashort cylinder of a plastic or like material, complete with a meshed oropen-worked bottom which is permeable to the reagent but which blocksthe passage of the specimens to be embedded.

The stack of box carriers 63 carrying the embedding boxes 62 of theforegoing construction are immovably captured between the pair of holderplates 60 and 61. For thus holding the stack of box carriers 63, thelower holder plate 61 has a pair of threaded studs 66 extending upwardlytherefrom and inserted in and through holes in the upper holder plate60. Nuts 67 are tightened on the top end portions of the threaded studs66 as they protrude from the holes in the upper holder plate 60.

Fluid passageways 68 and 69 are formed in and through the holder plates60 and 61, respectively, to establish communication between theinteriors of the embedding boxes 62 and the passageway 58 in the yoke 54as well as the passageway in the bottom fitting 53. It is to be notedthat despite the showing of FIGS. 6-8, the embedding boxes 62 and theircarriers 63 need not constitute separate units but can be of integralconstruction. Shown at 70 in FIGS. 6-8 is a thermostatic module of anyknown or suitable construction, preferably containing a thermoelectricrefrigerator working on the Peltier effect for heating and cooling thespecimens as well as the reagent that has been pumped into the embeddingboxes 62.

The following is a discussion of a method of embedding desired objectsof microscopic examination in a resin material by the micrologicalapparatus of the foregoing construction having the embedding boxes 62mounted to the embedding box holder 15. First, loaded with the embeddingboxes 62 containing the specimens to be embedded, the box carriers 63may be stacked up thereby providing a tubular arrangement of theembedding boxes 62 for the flow of a liquid therethrough. Then the stackof the loaded box carriers 63 may be mounted firmly between the pair ofholder plates 60 and 61 by tightening the nuts 67 on the studs 66. Thenthe stack of the loaded box carriers 63 with the holder plates 60 and 61may be mounted fast between the baseplate ledge 51 and the yoke 54 bytightening the nuts 56 on the studs 52. Now the mounting of theembedding boxes to the holder 15 has been completed.

Then the delivery pump 13 on the delivery conduit 12 may be set intooperation for delivering a selected reagent from either of thereceptacles 7a, 7b and 7c into the embedding boxes 62 in the holder 15by way of the delivery conduit 12, lower fitting 53 and passageway 69.The specimens within the embedding boxes 62 will be immersed in theselected reagent so delivered to the embedding box holder 15. Thedelivery pump 13 should be held out of operation during the impregnationof the specimens with the reagent.

During the time the delivery pump 13 is so held out of operation, thereagent select mechanism 9 may be driven for placing the deliveryconduit 12 in communication with the waste liquid conduit 25. Thereagent select mechanism 9 can be driven as, with the solenoid 30energized to raise the reciprocator 34 against the force of thecompression spring 35 and hence to disengage the plug 37 from the socketmember 38, the motor 27 is set into rotation for revolving the rotarypost 29. The motor 27 may be set out of rotation as the plug 37 reachesa position of vertical register with that one of the socket members 38which has the waste liquid conduit 25 coupled thereto. Then the solenoid30 may be de-energized to permit the plug 37 to move into fluid-tightengagement with the required socket member 38 under the bias of thecompression spring 35.

Also, while the delivery pump 13 is held out of operation as abovestated, the solenoid 42 of the shaker mechanism 14 may be excitedintermittently thereby imparting vibration to the reagent within theembedding boxes 62 and hence expediting the infiltration of the reagentinto the specimens. Each time the solenoid 42 is excited, the pair ofgripping jaws 44 will pivot against the forces of the compressionsprings 49, with the consequent constriction of the pliant hose 40 bythe pair of teeth 43. The constriction of the pliant hose 40 will causepart of the reagent on the downstream side of the shaker mechanism 14 toflow into the enlargement 16, which in this particular embodiment isformed by the fluid passageway 58 of relatively large cross section inthe yoke 54. Then, upon de-energization of the solenoid 42, the pair ofgripping jaws 44 will pivot away from each other owing to the forces ofthe compression springs 49. The hose 40 will then expand by virtue ofits own elasticity to cause reverse flow of the reagent from itsdownstream side. The repeated energization and de-energization of thesolenoid 42 at fine intervals will lead to the ready permeation of thereagent through the specimens.

Following the shaking of the reagent for a preassigned length of timeand, consequently, the complete impregnation of the specimens with thereagent, the delivery pump 13 may be set into rotation in a reversedirection for the discharge of the excess reagent into the wastereceptacle 18. For such discharge of the used reagent the three-wayvalve 19 should be actuated to place the waste receptacle 18 incommunication with the air filter 21. Air will then be delivered to theembedding boxes 15 by way of the overflow conduit 17 for smoothdischarge of the used reagent.

The vacuum pump 22 provided to the waste receptacle 18 serves thepurpose of evacuating the embedding box holder 15 as when the specimenswithin the embedding boses are being treated with glutaraldehyde orembedded in a resin. The exhaust gases produced during the operation ofthe vacuum pump 22 are cleaned as they pass through the activatedcharcoal filter 24.

Upon completion of the discharge of the used reagent, the reagent selectmechanism 9 may be re-actuated for placing the delivery conduit 12 incommunication with some other of the reagent receptacles 7a, 7b or 7ccontaining another desired reagent. This second selected reagent will befed into the embedding boxes 62 as the delivery pump 13 is again setinto rotation in a forward direction.

The foregoing cycle of operation may be repeated for the embedment ofthe specimens within the embedding boxes 62. For treating the specimenswith an alcohol of progressively greater concentrations, a lowerconcentration (e.g. 50%) alcohol may first be supplied to the embeddingboxes 62. Then, with this lower concentration alcohol left undischarged,a higher concentration (e.g. 100%) alcohol may be supplied to theembedding boxes 62. The alcohol concentration within the embedding boxes62 will become gradually higher as the excess amount is dischargedthrough the overflow conduit 17.

Such being the construction and operation of the apparatus in accordancewith the invention, it will be appreciated that only minimal amounts ofreagents are required for the embodment of the specimens, and therunning costs of the apparatus will also be reduced to a minimum.Further, instead of dipping the specimens in the reagents, the reagentsare pumped into the embedding boxes in their holder in accordance withthe invention. This makes it possible to treat the specimens with analcohol of various concentrations merely by intermingling two alcoholpreparations of 50% and 100% concentrations which are availablecommercially, without the need for the provision of alcohol preparationsof many different concentrations. Still further, the specimens will befavorably embedded as the reagents and resin are positively fed into theembedding boxes.

INDUSTRIAL APPLICABILITY

Objects of microscopic examination can be efficiently embedded in aresin or like material, so that the invention finds application inmedical and scientific fields for the examination of living tissues orthe like.

I claim:
 1. Apparatus for the preparation of objects for microscopicexamination comprising a reagent housing, a plurality of reagentreceptacles placed in said housing for containing different reagents fortreating specimens, a delivery pump capable of bidirectional rotationand of blocking the flow of a reagent therethrough when out ofoperation, a reagent select mechanism connected between said reagenthousing and said delivery pump for placing any selected one of saidreagent receptacles in communication with said delivery pump, anembedding box holder for holding in a stack a plurality of embeddingboxes containing specimens for microscopic examination, said deliverypump being connected to said embedding box holder for causing anyselected reagent to flow through said embedding boxes, and a reagentshaker mechanism, provided between said embedding box holder and saiddelivery pump, said shaker mechanism having means for causing repeatedforward and backward vertical flows through said embedding box holder,at closely spaced intervals, of the selected reagent in the embeddingbox holder while the delivery pump is out of operation.
 2. Apparatus forthe preparation of objects for microscopic examination as claimed inclaim 1, wherein said embedding box holder is elongated vertically forholding a vertical stack of embedding boxes.
 3. Apparatus for thepreparation of objects for microscopic examination as claimed in claim1, wherein said embedding box holder comprises an upstanding baseplateholding a plurality of annular box carriers fluid-tightly stacked up,each box carrier having mounted thereto one of the embedding boxes, andholder means for holding the stack of box carriers.
 4. Apparatus for thepreparation of objects for microscopic examination as claimed in claim1, wherein said reagent select mechanism comprises a table having aplurality of socket members communicating with said separate reagentreceptacles via suction conduits, a rotary post rotatably mounted onsaid table, and plug reciprocator means for vertically reciprocating aplug into and out of selective engagement with said socket members, saidplug communicating with said delivery pump via a delivery conduit. 5.Apparatus for the preparation of objects for microscopic examination asclaimed in claim 1, wherein said embedding box holder communicates witha waste receptacle via an overflow conduit having a portion of enlargedcross section, said waste receptacle communicating with one of socketmembers of said reagent select mechanism via a waste liquid conduit. 6.Apparatus for the preparation of objects for microscopic examinationcomprising an embedding box holder, a stack of embedding boxes containedin said holder and each containing a specimen, a pump for selectivelydelivering reagents to said stack of embedding boxes, a reagent shakermechanism disposed under said stack of embedding boxes for vibrating areagent within the embedding box holder holding said stack of embeddingboxes, said reagent shaker mechanism comprising a pair of gripping jawsfor cyclically gripping and releasing a conduit through which saidreagents are fed to said stack of embedding boxes, and drive means fordriving said pair of gripping jaws.
 7. Apparatus for the preparation ofobjects for microscopic examination as claimed in claim 6, wherein saidembedding box holder includes an upstanding baseplate holding aplurality of annular box carriers fluid-tightly stacked up, each boxcarrier having mounted thereto one of the embedding boxes, and holdermeans for holding the stack of box carrier.
 8. Apparatus for thepreparation of objects for microscopic examination as claimed in claim6, wherein said apparatus further comprises a reagent select mechanismfor selectively supplying the reagents to said stack of embedding boxes,said reagent select mechanism comprising a table having a plurality ofsocket members communicating with separate reagent receptacles viasuction conduits, a rotary post rotatably mounted on said table, andplug reciprocator means for vertically reciprocating a plug into and outof selective engagement with said socket members, said plugcommunicating with said delivery pump via a delivery conduit. 9.Apparatus for the preparation of objects for microscopic examinationcomprising a reagent housing, a plurality of reagent receptacles placedin said housing for containing different reagents for treatingspecimens, a delivery pump capable of bidirectional rotation and ofblocking the flow of a reagent therethrough when out of operation, areagent select mechanism connected between said reagent housing and saiddelivery pump for placing any selected one of said reagent receptaclesin communication with said delivery pump, a vertically elongatedembedding box holder for holding in a stack a plurality of embeddingboxes containing specimens for microscopic examination, said deliverypump communicating with said embedding box holder via an elastic conduitfor causing any selected reagent to flow through said embedding boxes,and a reagent shaker mechanism provided under said embedding box holderfor shaking the reagent within said embedding box holder, said reagentshaker mechanism comprising a pair of gripping jaws for cyclicallygripping and releasing said elastic conduit, and drive means for drivingsaid pair of gripping jaws.